Nucleic acid probes and methods for detecting plasmodium parasites

ABSTRACT

This invention relates to novel nucleic acid-based probes and methods for detecting  Plasmodium  parasites in biological samples as well as detecting different  Plasmodium  parasites selectively from one another.

BACKGROUND OF THE INVENTION

Plasmodium is a genus of parasitic protozoa. Infection with this genusis known as malaria. The parasite always has two hosts in its lifecycle: a mosquito vector and a vertebrate host. At least ten speciesinfect humans including P. falciparum, P. vivax, P. malariae, and P.ovale. Malaria is an infectious disease that is widespread in tropicaland subtropical regions. Malaria represents a threat to survival of men,women and children. It infects between 300 and 500 million people everyyear and causes between one and three million deaths annually, mostlyamong young children in Sub-Saharan Africa.

Malaria is one of the most common infectious diseases and an enormouspublic-health problem. The disease is caused by protozoan parasites ofthe genus Plasmodium. The most serious forms of the disease are causedby Plasmodium falciparum and Plasmodium vivax, but other related species(Plasmodium ovale and Plasmodium malariae) can also infect humans.Determining the infectious species can help determine the course oftreatment for the patient.

The preferred and most reliable diagnosis of malaria is microscopicexamination of blood films because each of the four major parasitespecies has distinguishing characteristics. Two sorts of blood film aretraditionally used. Thin films are similar to usual blood films andallow species identification because the parasite's appearance is bestpreserved in this preparation. Thick films allow the microscopist toscreen a larger volume of blood and are about eleven times moresensitive than the thin film, so picking up low levels of infection iseasier on the thick film, but the appearance of the parasite is muchmore distorted and therefore distinguishing between the differentspecies can be much more difficult.

From the thick film, an experienced microscopist can detect parasitelevels (or parasitemia) down to as low as 0.0000001% of red blood cells.However, microscopic diagnosis can be difficult because the earlytrophozoites (“ring form”) of all four species look identical and it isnever possible to diagnose species on the basis of a single ring form;species identification is always based on several trophozoites.

In areas where microscopy is not available, there are antigen detectiontests that require only a drop of blood. OptiMAL-IT® (TCS Bio Sciences,Buckingham, England) will reliably detect falciparum down to 0.01%parasitemia and non-falciparum down to 0.1%. Paracheck-Pf® (OrchardBiomedical Systems, India) will detect parasitemias down to 0.002% butwill not distinguish between falciparum and non-falciparum malaria.Parasite nucleic acids may also be detected using polymerase chainreaction. This technique is more accurate than microscopy. However, itis expensive and requires a specialized laboratory. Moreover, levels ofparasitemia are not necessarily correlative with the progression ofdisease, particularly when the parasite is able to adhere to bloodvessel walls. Limited molecular methods are available in some clinicallaboratories and rapid real-time assays, for example, QT-NASBA(real-time quantitative nucleic acid sequence-based amplification) basedon the polymerase chain reaction) but are only now being developed.Therefore, sensitive, low-tech diagnosis tools need to be developed forin order to detect low levels of parasitaemia in the field.

What is need are reagents and methods for the rapid and accuratedetection and discrimination of various malaria causing Plasmodiumspecies.

SUMMARY OF THE INVENTION

This invention relates to nucleic acid probes that detect anddiscriminate between different species of Plasmodium parasites in, forexample, hybridization assays. Accordingly, in a first aspect, theinvention features nucleic acid fragments to be used as probes fordetecting Plasmodium in a hybridization assay. The invention alsoincludes probes (DNA, RNA and PNA) that can discriminate between P.falciparum, P. vivax, P. malariae and P. ovale. In the context of thepresent invention the term “discriminates between” (or similar terms)means that the probe binds to nucleic acid (e.g., RNA, DNA, rRNA[ribosomal RNA] or rDNA [ribosomal DNA]) from one species more favorablythan the other 3 species.

The probes of the present invention are able to detect specific speciesof Plasmodium in a sample without cross-reacting with other species ofPlasmodium to any significant extent. In this regard, cross-reactivitybetween the probes of the present invention in the detection of variousPlasmodium species is less than about 20%, less than about 15%, lessthan about 10%, less than about 5% or less than about 2%.

In a another aspect, the invention features a nucleic acid fragmentcontaining a sequence selected from at least five, preferably, at leastten, more preferably at least thirteen or most preferably at leastfifteen consecutive nucleotides or the entire sequence of one or more ofprobes designated SEQ ID NO: 1 through SEQ ID NO.: 4 or the partial orfull-length complementary sequences thereof.

In a another aspect, the invention features a method for detecting thepresence of Plasmodium in a sample. In this method, a sample iscontacted with a nucleic acid fragment containing a sequence selectedfrom, preferably, at least five, at least ten, more preferably at leastthirteen or most preferably at least fifteen consecutive nucleotides orthe entire sequence of a PV1, PF4, PM1 or PO1, probe selected from orthe partial or full-length complementary sequence thereof (or anycombination thereof); under conditions that permit the nucleic acidfragment to hybridize to Plasmodium nucleic acid. Detection of thenucleic acid fragment bound to the Plasmodium nucleic acid in the sampleis used as an indication of the presence of Plasmodium in the sample.Detection with probes of the present invention that discriminate betweenthe four species of Plasmodium listed above indicates the presence ofthat species of Plasmodium.

In specific aspects, the present invention contemplates a method fordetecting the presence of Plasmodium in a sample, said method comprisingthe steps of: contacting said sample with a probe for detectingPlasmodium in a hybridization assay, wherein said probe discriminatesbetween Plasmodium species, under conditions that permit said probe tohybridize to Plasmodium nucleic acid; and detecting said probe bound tosaid Plasmodium nucleic acid in said sample as an indication of thepresence of Plasmodium in said sample.

In the preceding embodiment, the nucleic acid fragment comprises asequence that is selected from at least five consecutive nucleotides ofprobe PV1, PF4, PM1 or PO1 the full-length complementary sequencethereof.

The present invention also contemplates a nucleic acid fragmentcomprising a sequence selected from at least ten (10), at least thirteen(13) or at least fifteen (15) consecutive nucleotides of a probeselected from a group consisting of PV1, PF4, PM1, PO1 or thefull-length complementary sequence thereof.

The present invention also contemplates a nucleic acid fragmentcomprising the complete sequence selected of a probe selected from agroup consisting of PV1, PF4, PM1, PO1 or the full-length complementarysequence thereof.

In specific aspects, the present invention contemplates a method ofselecting nucleic acid probes that discriminate between the species P.falciparum, P. vivax, P. malariae and P. ovale, the method comprising:preparing a nucleic acid fragment or polypeptide nucleic acid (PNA)corresponding to, or complementary to, a sequence of at least fivenucleotides of nucleic acid from P. falciparum, P. vivax, P. malariaeand P. ovale; comparing the ability of the probe to detect one or moreof the Plasmodium species in a hybridization assay; and selecting theprobe or probes that detect one, two or three species of Plasmodium butnot all four species of Plasmodium.

The present invention also contemplates a method for detecting anddifferentiating between P. falciparum, P. Vivax, P. malariae and P.ovale in a sample, said method comprising: providing: i) a sample from,for example, a subject suspected of having malaria and ii) probescomprising nucleic acid suitable for detecting and differentiatingbetween P. falciparum, P. Vivax, P. malariae and P. ovale; contactingsaid sample with said probes under conditions suitable for hybridizationof said probes to targets; and determining the presence of P.falciparum, P. Vivax, P. malariae and P. ovale, if any, in the sample.

In any of the preceding embodiments, the probe or probes used may be ofthe entire nucleotide sequence as disclosed herein or the complementarystrand thereof as well as the complementary sequence of the at leastfive (5), at least ten (10), at least thirteen (13) or at least fifteen(15) contiguous nucleotides of the probes.

DETAILED DESCRIPTION OF THE INVENTION

The invention features nucleic acid probes for detecting Plasmodiumparasites (e.g., P. falciaprum, P. vivax, P. malariae and P. ovale) in,for example, hybridization assays. The probes of the invention may beused in methods for detecting the presence of Plasmodium in a biologicalsample. In these methods, a probe of the invention is contacted with abiological sample or specimen (e.g., whole blood, cerebrospinal fluid(CSF), a tissue sample or any other biological sample suspected ofhaving the presence of Plasmodium) in a hybridization assay anddetection of the probe bound to the nucleic acid in the sample is usedas an indication of the presence of Plasmodium in the sample.

Probes included in the invention may be identified by:

A (1) preparing a nucleic acid fragment (DNA, RNA) or polypeptidenucleic acid (PNA) (i.e., a probe) corresponding to or complementary to,a sequence of at least five, at least ten, at least thirteen or at leastfifteen nucleotides of nucleic acid from P. falciparum and (2) comparingthe ability of the probe to detect all the Plasmodium species in ahybridization assay. Probes that hybridize to P. falciparum morefavorably than to other three species are included in the invention.

B (1) preparing a nucleic acid fragment (DNA, RNA) or PNA (i.e., aprobe) corresponding to, or complementary to, a sequence of at leastfive, at least ten, at least thirteen or at least fifteen nucleotidesfrom P. vivax and (2) comparing the ability of the probe to detect allthe Plasmodium species in a hybridization assay. Probes that hybridizeto P. vivax more favorably than to other three species are included inthe invention.

C (1) preparing a nucleic acid fragment (DNA, RNA) or PNA (i.e., aprobe) corresponding to, or complementary to, a sequence of at leastfive, at least ten, at least thirteen or at least fifteen nucleotidesfrom P. malariae and (2) comparing the ability of the probe to detectall the Plasmodium species in a hybridization assay. Probes thathybridize to P. malariae more favorably than to other three species areincluded in the invention.

D (1) preparing a nucleic acid fragment (DNA, RNA) or PNA (i.e., aprobe) corresponding to, or complementary to, a sequence of at leastfive, at least ten, at least thirteen or at least fifteen nucleotidesfrom P. ovale and (2) comparing the ability of the probe to detect allthe Plasmodium species in a hybridization assay. Probes that hybridizeto P. ovale more favorably than to other three species are included inthe invention.

P. falciaprum, P. vivax, P. malariae and P. ovale nucleic acid may beobtained from biological samples (such as whole blood, bone marrow, CSF)from infected individuals, using standard nucleic acid isolation methodsin the art. P. falciaprum (as well as P. vivax, P. malariae and P.ovale) can also be obtained from culture. For example, DNA encodingPlasmodium ribosomal RNA may be obtained by PCR amplification of DNAprepared from a whole blood sample of an infected patient using themethods and primers described herein and known in the art.

Any Plasmodium sequence (e.g., a sequence encoding 58, 5.8S, 18S, or 28Sribosomal RNA) may be selected as a candidate sequence for theidentification of probes. Preferred sequences are those that divergefrom analogous sequences in non-human Plasmodium or other protozoanparasites like, for example, Babesia or Thileria, as determined byphylogenetic comparison. The nucleic acid probes of the invention are atleast 5, at least 10, at least 13 or at least 15 nucleotides in lengthand may contain deoxyribonucleotides (DNA probes), ribonucleotides (RNAprobes), peptide nucleic acid (PNA probes) or combinations ormodifications thereof. The probes may be single stranded or doublestranded and may be prepared by any of a number of standard methods inthe art. For example, the probes may be made by chemical synthesis,restriction endonuclease digestion of a vector (e.g., a plasmidcontaining a sequence corresponding to the probe), polymerase chainreaction (PCR) amplification, or in vitro transcription of a vectorcontaining a sequence corresponding to the probe (see, e.g., Ausubel, etal., Current Protocols in Molecular Biology, Greene Publishing, NewYork, N.Y., 1994, incorporated herein by reference). The probes may belabeled during or after synthesis. For example, labeled nucleotidescontaining, e.g., radioisotopes (e.g., p32, S35, or H3), biotin ordigoxigenin may be incorporated into the probe during synthesis. Probescontaining biotin are detected by the use of a secondary reagent such asavidin or streptavidin, which contains a detectable label such as afluorochrome (e.g., fluorescein or rhodamine) or an enzyme (e.g.,alkaline phosphatase or horseradish peroxidase). Similarly, probescontaining digoxigenin may be detected by using a labeledantidigoxigenin antibody. Probes may also be labeled after synthesis by,e.g., nick translation or the use of T4 RNA ligase, poly(A) polymerase,terminal transferase or T4 polynucleotide kinase, in standard methods(see, e.g., Ausubel, et al., supra). The probes may also containmodified nucleotides in order to increase the stability of the probe.For example, ribonucleotides containing 2′-0-alkyl groups on the ribosegroup may be used. The probes may also contain modifications thatfacilitate capture of the probe onto a solid support. For example,poly-dA or poly-deaza-guanosine tails may be added to the 3′ ends of theprobes, using terminal transferase, in order to facilitate probe bindingto a solid support, e.g., poly-dT or poly-dC labeled magnetic particles.The probes may be purified prior to use, using standard methods such asdenaturing polyacrylamide gel electrophoresis, high performance liquidchromatography or gel filtration chromatography (see, e.g., Ausubel, etal., supra). The probes of the invention may be used in any standardhybridization assay to detect the presence of Plasmodium in a sample.For example, Southern blot, dot blot, in situ hybridization, real-timehybridization detection by biosensors or dual probe, sandwich-typehybridization assays may be used (see, e.g., U.S. patent applicationSer. No. 07/826,657 [now U.S. Pat. No. 5,519,127] and U.S. Pat. No.5,629,156 [International Publication Number WO 94/10335], all of whichare incorporated herein by reference). Alternatively, the probes may beused as primers in a polymerase chain reaction assay (see, e.g.,Ausubel, et al., supra). Biological samples that may be analyzed usingthe probes and methods of the invention include whole blood, CSF, bonemarrow and tissue samples from, e.g., the spleen. Nucleic acid isextracted from the sample by standard methods (except in the case of insitu hybridization, where the cells are kept intact) and is analyzedusing the probes in the assays listed above. A single probe orcombinations of probes may be used in the assay. The hybridizationconditions used with the probes (e.g., in the methods of the invention)fall within the range of, for example, about 30 to about 50% formamideat about 25° C. to about 42° C. or mixtures of GuSCN and formamide atabout 25° C. to about 37° C. As is “known by one skilled in the art,”selection of hybridization conditions depends on the length andnucleotide content (i.e., GC compared to AT) of the probe. Accordingly,hybridization conditions may be adjusted to accommodate these factors.In addition, the use of different salts (e.g., guanidine thiocyanate orguanidine hydrochoride compared with NaCl) and denaturing agents (e.g.,NP-40, sodium dodecyl sulfate) may require adjustment of the saltconcentration and the temperature, as can readily be determined by oneskilled in the art.

Non-limiting examples of hybridization conditions that may be used inthe present invention are as follows. In Southern blot analysis, thefollowing hybridization conditions may be used: 30% to 50% formamide in2×SSC at about 42° C. After hybridization, the filters are washed usingstandard methods. For example, three 15 minute post-hybridization washesat about 25° C. in 2×SSC to 0.1×SSC and 0.1% SDS may be carried out inorder to remove unbound probes. For RNA blots hybridizations in about30% formamide at room temperature overnight were performed. Excessprobes were removed by washing three 15 min washes in 2×SSC with 0.1%SDS.

The term “hybridization” refers to the pairing of complementary nucleicacids. Hybridization and the strength of hybridization (i.e., thestrength of the association between the nucleic acids) is impacted bysuch factors as the degree of complementary between the nucleic acids,stringency of the conditions involved, the T_(m) of the formed hybrid,and the G-C ratio within the nucleic acids. A single molecule thatcontains pairing of complementary nucleic acids within its structure issaid to be “self-hybridized.”

It is well known that numerous equivalent conditions may be employed tocomprise suitable hybridization conditions; factors such as the lengthand nature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.).

For in situ hybridization, the following conditions may be used asdescribed in U.S. Pat. No. 6,165,723 and U.S. patent application Ser.No. 11/494,430 (which are herein incorporated by reference): GuSCN (1.5to 3.5 M depending on the probe sequence) between room temperature andabout 37° C. or mixtures of GuSCN and formamide between room temperatureand about 37° C. for 30 minutes to one hour, followed by washes in SSC(2× to 0.1×) and 0.1% SDS.

Fluorescent markers (dyes) suitable for use in the present inventioninclude, but are not limited to, for example, Alexa 488, rhodamin, Texasred, fluorescein, Oregon Green, Tokyo Green as well as others known tothose of ordinary skill in the field at the time of the invention.

EXEMPLIFICATION

Without further elaboration, it is believed that one skilled in the artcan, based on the description herein, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative and not limitative of theremainder of the disclosure in any way whatsoever.

Hybridization of Probes PF4, PV1, PM1 and PO1 to P. falciaprum, P.vivax, P. ovale and P. malariae Samples, Respectively.

Dot-blot analysis data is shown in Table 1. Dot-blot protocol andanalysis is known in the art. See, for example, Brown, T., Dot and SlotBlotting DNA, Curr. Protoc. Mol. Biol., 2001 May; Chapter 2:Unit2.9B andreferences therein. In the present experiments, approximately 0.1 ug ofa plasmid containing nucleotide sequences encoding the respective 18SrRNA subunits was used per spot. The blots were hybridized withdig-labeled probes under the hybridization conditions described above.

In another embodiment, fluorescent in situ hybridization (FISH) may beused to detect the probe(s) of the present invention. FISH is well knownto those of skill in the art. For example, see, Volpi, and Bridger, FISHglossary: An Overview of the Fluorescence in situ HybridizationTechnique, Biotechniques, 2008 Oct;45(4):385-6, 388, 390 passim, andreferences therein.

In the present experiment, a dot blot assay was employed. In the case ofRNA, RNA was synthesized and 0.1 ug in 6×SSC was spotted onnitrocellulose membrane. Hybridization with the dig-labeled probes(digoxigenin-labeled probes) was performed overnight at room temperaturein formamide. This method was used to compare hybridization signalsbetween the different organisms and probes.

Plasmodium Species 18S rRNA-specific Probes

Plasmodium species specific probes of the invention include probes whichhave the sequences of a probe selected from PV1, PF4, PM1 and PO1 or thefull-length complementary sequence thereof.

P. vivax Specific Probe [SEQ ID NO.: 1] PV15′-AGCAAAATGCGCACAAAGTCGATACGAAGTATC-3′ P. falciparum Specific Probe[SEQ ID NO.: 2] PF4 5′-TTACAAAACCAAAAATTGGCCTTGCATTGTTATTT-3′ P.malariae Specific Probe [SEQ ID NO.: 3] PM15′-GAAACACTCATATATAAGAATGTCTC-3′ P. ovale Specific Probe [SEQ ID NO.: 4]PO1 5′-AATTTCCCCGAAAGGAATTTTC-3′

TABLE 1 Probes P. falciparum P. vivax P. malariae P. ovale PV1 NegativePositive Negative Negative PF4 Positive Negative Negative Negative PM1Negative Negative Positive Negative PO1 Negative Negative NegativePositive

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

1. A method for detecting the presence of Plasmodium falciparum in asample, said method comprising the steps of: a) contacting said samplewith a nucleic acid probe suitable for detecting Plasmodium falciparumin a hybridization assay, the probe consisting of [SEQ ID NO.: 2] underconditions that permit said probe to hybridize to Plasmodium falciparumnucleic acid; and b) detecting said probe bound to said Plasmodiumfalciparum nucleic acid in said sample as an indication of the presenceof Plasmodium falciparum in said sample.
 2. The method of claim 1,wherein said nucleic acid probe is a sequence that is selected from atleast five consecutive nucleotides of [SEQ ID NO.: 2] or the full-lengthcomplementary sequence thereof.
 3. A method for detecting the presenceof Plasmodium vivax in a sample, said method comprising the steps of: a)contacting said sample with a probe fsuitable for detecting Plasmodiumvivax in a hybridization assay, the probe consisting of [SEQ ID NO.: 1]under conditions that permit said probe to hybridize to Plasmodium vivaxnucleic acid; and b) detecting said probe bound to said Plasmodium vivaxnucleic acid in said sample as an indication of the presence ofPlasmodium vivax in said sample.
 4. The method of claim 3, wherein saidnucleic acid probe is a sequence that is selected from at least fiveconsecutive nucleotides of [SEQ ID NO.: 1] or the full-lengthcomplementary sequence thereof.
 5. A method for detecting the presenceof Plasmodium ovale in a sample, said method comprising the steps of: a)contacting said sample with a probe suitable for detecting Plasmodiumovale in a hybridization assay, the probe consisting of [SEQ ID NO.: 4]under conditions that permit said probe to hybridize to Plasmodium ovalenucleic acid; and b) detecting said probe bound to said Plasmodium ovalenucleic acid in said sample as an indication of the presence ofPlasmodium ovale in said sample.
 6. The method of claim 5, wherein saidnucleic acid probe is a sequence that is selected from at least fiveconsecutive nucleotides of [SEQ ID NO.: 4] or the full-lengthcomplementary sequence thereof.
 7. A method for detecting the presenceof Plasmodium malariae in a sample, said method comprising the steps of:a) contacting said sample with a probe suitable for detecting Plasmodiummalariae in a hybridization assay, the probe consisting of [SEQ ID NO.:3] under conditions that permit said probe to hybridize to Plasmodium omalariae vale nucleic acid; and b) detecting said probe bound to saidPlasmodium malariae nucleic acid in said sample as an indication of thepresence of Plasmodium malariae in said sample.
 8. The method of claim7, wherein said nucleic acid probe is a sequence that is selected fromat least five consecutive nucleotides of [SEQ ID NO.: 3] or thefull-length complementary sequence thereof.
 9. The method of claim 1, 3,5 or 7 whereby the detection method used is Fluorescent in SituHybridization (FISH).
 10. The method of claim 1, 3, 5 or 7 whereby thedetection method used is a Dot Blot assay.
 11. A method of selectingnucleic acid probes that discriminate between the species P. faciparum,P. vivax, P. malariae and P. ovale, the method comprising: a) preparinga nucleic acid fragment or polypeptide nucleic acid, PNA correspondingto, or complementary to, a sequence of at least five nucleotides ofnucleic acid from P. faciparum, P. vivax, P. malariae and P. ovale; b)comparing the ability of the probe to detect one or more of thePlasmodium species in a hybridization assay; and c) selecting the probeor probes that detect one, two or three species of Plasmodium but notall four species of Plasmodium.